WO2021028359A1

WO2021028359A1 - Controlled expression of chimeric antigen receptors in t cells

Info

Publication number: WO2021028359A1
Application number: PCT/EP2020/072303
Authority: WO
Inventors: David FENARD; Tobias Abel
Original assignee: Sangamo Therapeutics France
Priority date: 2019-08-09
Filing date: 2020-08-07
Publication date: 2021-02-18

Abstract

The present disclosure provides expression constructs comprising a FOXP3- derived promoter and T cells comprising the expression constructs. Also provided are methods of generating the cells and methods of using the cells to treat patients in need of modulation of an immune response, such as, for example, patients with a disease or disorder selected from the group consisting of an inflammatory disease or condition, an autoimmune disease or condition, an allergic disease or condition, or an organ transplantation condition, or having received or planned to receive a tissue transplantation or patients with cancer or with an infectious disease.

Description

CONTROLLED EXPRESSION OF CHIMERIC ANTIGEN RECEPTORS IN

T CELLS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Application 62/884,914, filed August 9, 2019, the disclosure of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 31, 2020, is named Sequence listing.txt and is 67,135 bytes in size.

BACKGROUND OF THE INVENTION

A healthy immune system is one that is in balance. Cells involved in adaptive immunity include B and T lymphocytes. There are two general types of T lymphocytes - effector T (Teff) cells and regulatory T (Treg) cells. Teff cells include CD4⁺ T helper cells and CD8⁺ cytotoxic T cells. Teff cells play a central role in cellular-mediated immunity following antigen challenge. A key regulator of the Teff cells and other immune cells is the Treg cells, which prevent excessive immune responses and autoimmunity (see, e.g., Romano et al., Front Imm. (2019) 10, art. 43).

Some Tregs are generated in the thymus; they are known as natural Treg (nTreg) or thymic Treg (tTreg). Other Tregs are generated in the periphery following antigen encounter or in cell culture, and are known as induced Tregs (iTreg) or adaptive Tregs. Tregs actively control the proliferation and activation of other immune cells, including inducing tolerance, through cell-to-cell contact involving specific cell surface receptors and the secretion of inhibitory cytokines such as IL-10, TGF-b and IL-35 (Dominguez-Villar and Hafler, Nat Immunol. (2018) 19: 665-673). Failure of tolerance can lead to autoimmunity and chronic inflammation. Loss of tolerance can be caused by defects in Treg functions or insufficient Treg numbers, or by unresponsive or over-activated Teff (Sadi on et al, Clin Transllmm. (2018) 7: el Oil, doi:10-1002/cti2.1011).

In recent years, there has been much interest in the use of Tregs to treat diseases. A number of approaches, including adoptive cell therapy, have been explored to boost Treg numbers and functions in order to treat autoimmune diseases. Treg transfer (delivering an activated and expanded population of Tregs) has been tested in patients with autoimmune diseases such as type I diabetes, cutaneous lupus erythematosus, and Crohn’s disease (Dominguez-Villar, supra). However, these cell populations are polyclonal in nature and thus may not be as effective as hoped. There also is evidence that simply increasing the number of Tregs may not be sufficient to control disease (McGovern et al., Front Imm. (2017) 8, art. 1517).

Thus, there remains a need for effective cell therapy that can treat diseases associated with unwanted proliferation and activation of Teff cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a nucleic acid expression construct, comprising a coding sequence for a protein of interest that is not FOXP3, wherein the coding sequence is operably linked to a promoter derived from a human FOXP3 gene, said promoter comprising nucleotide -769 to nucleotide -729 (SEQ ID NO: 5) of a human FOXP3 gene.

In some embodiments, the promoter comprises nucleotide -900 to nucleotide +3 (SEQ ID NO: 1), nucleotide -900 to nucleotide +172 (SEQ ID NO: 2), nucleotide -1,799 to nucleotide +3 (SEQ ID NO: 3), or nucleotide -1,799 to nucleotide +172 (SEQ ID NO: 4) of the human FOXP3 gene. In certain embodiments, the nucleic acid expression construct is a lentiviral construct, an adenoviral construct, an adeno-associated viral construct, a plasmid, a DNA construct, or an RNA construct.

In some embodiments, the protein of interest expressed by the construct is a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR) or a T-cell receptor (TCR). In further embodiments, the CAR or TCR is specific for (i) an autoantigen, (ii) a B cell antigen optionally selected from CD 19 and CD20, (iii) an allogeneic HLA class I molecule, wherein the class I molecule is optionally HLA-A2, (iv) an antigen involved in a disease, or (v) an antigen present or expressed in a specific tissue or organ, or present or expressed at a site of inflammation.

In further embodiments, the CAR or TCR is specific for an antigen involved in a disease. In further embodiments, the CAR or TCR is specific for an antigen present or expressed in a specific tissue or organ, or present or expressed at a site of inflammation.

In another aspect, the present disclosure provides a genetically engineered mammalian cell comprising one or more of the present nucleic acid expression constructs. The cell may be, without limitation, a lymphoid cell, a lymphoid progenitor cell, a mesenchymal stem cell, a hematopoietic stem cell, an induced pluripotent stem cell, or an embryonic stem cell. In particular embodiments, the cell is a T cell (e.g., a Teff or Treg cell), a natural killer (NK) cell, or another immune cell.

In some embodiments, the genetically engineered cell comprises a null mutation in a gene selected from a T cell receptor alpha or beta chain gene, a HLA Class I or II gene, a HLA Class II regulator gene (e.g., RFXANK (regulatory factor X associated Ankyrin containing protein), RFX5 (regulatory factor X5), RFXAP (regulatory factor X associated protein; RFX subunits), and CIITA (Class II major histocompatibility complex transactivator)), a transporter associated with antigen processing, a minor histocompatibility antigen gene, and a b2 microglobulin (B2M) gene. In particular embodiments, the engineered cells of the present disclosure are human cells. In further embodiments, the present genetically engineered cell comprises a suicide gene optionally selected from a HSV-TK gene, a cytosine deaminase gene, a nitroreductase gene, a cytochrome P450 gene, or a caspase-9 gene.

In another aspect, the present disclosure provides a method of treating a patient in need of immunosuppression, comprising administering to the patient the present genetically engineered Treg cells; use of the present Treg cells in the manufacture of a medicament in treating a patient in need of immunosuppression; and the present genetically engineered Treg cells for use in treating a patient in need of immunosuppression. In some embodiments, the patient has a disease or disorder such as an inflammatory disease, an autoimmune disease, an allergic disease, or a condition associated with organ transplantation (e.g., graft-versus-host disease). In some embodiments, the patient has an autoimmune disease, or has received or will receive tissue transplantation. In some embodiments, the patient has a condition associated with an organ transplantation. In some embodiments, the patient has an inflammatory disease. In some embodiments, the patient has an autoimmune disease. In some embodiments, the patient has an allergic disease.

In another aspect, the present disclosure provides a method of treating cancer in a patient, comprising administering to the patient the present genetically engineered Teff cell; use of the present Teff cells in the manufacture of a medicament in treating cancer in a patient; and the present engineered Teff cells for use in treating cancer in a patient.

In another aspect, the present disclosure provides a method of treating an infectious disease in a patient, comprising administering to the patient the present genetically engineered Teff cell; use of the present Teff cells in the manufacture of a medicament in treating an infectious disease in a patient; and the present engineered Teff cells for use in treating an infectious disease in a patient.

In some embodiments, the above methods and use are for treating human patients and the engineered cells are autologous or allogeneic human cells. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the following four CAR constructs on the top panel: (i) HLA-A2 (i.e., HLA.A2) CAR under the control of the human phosphoglycerate kinase (PGK) promoter (pTX135); (ii) the construct of (i) with the addition of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Mutant 6 (WpreMut6) regulatory region (pTX136); (iii) HLA-A2 CAR under the control of the human elongation factor 1 alpha (EFla) promoter (pTX137); and (iv) the construct of (iii) with the addition of the WpreMut6 regulatory region (pTX138). On the bottom panel, flow cytometry graphs show the CAR expression level on the surface of Treg cells transduced with the TX135, TX136, TX137, or TX138 lentiviral vector. Transduction efficiency (%) was assessed using flow cytometry after the cells were stained with APC-labeled HLA-A2 dextramer. UT: un- transduced.

FIG. 2 shows FACS plots of CAR-positive Tregs on the top panel, where the expression levels of the CAR are indicated as low, medium, or high. The bottom panel is a histogram graph showing the percentage of low-, medium-, and high-expressing Tregs for each CAR expression cassette (TX135 to TX138). SSC: side scatter.

FIG. 3 is a graph monitoring the Treg phenotype at the end of the first cycle of expansion. Treg cells were labeled with antibodies directed against human CD4, CD25, CD 127, and CTLA-4. For detection of FOXP3 and Helios transcription factors, an intra-nuclear labeling was performed. From left to right: untransduced cells (UT); and cells transduced with TX135, TX136, TX137, and TX138, respectively.

FIG. 4 shows human Treg activation following CAR interaction with HFA-A2. Panel A: Treg cells transduced with TX135 were incubated with anti-CD3/CD28 beads, HPA-A2 dextramer, and HPA-A2 negative (HPA.A2 neg) and positive (HPA.A2 pos) peripheral blood mononuclear cells (PBMCs); after 24 hours, the cell surface expression of CD69 was monitored using flow cytometry. Panel B: Treg cells untransduced (UT) or transduced with TX135, TX136, TX137 or TX138 were not stimulated (None) or stimulated as in Panel A (i.e., with anti-CD3/CD28 beads (Beads), HLA-A2 dextramer (Dex), or HLA-A2 negative (A2neg) or positive (A2pos) peripheral blood mononuclear cells (PBMCs)). From left to right: UT, TX135, TX136, TX137, and TX138.

FIG. 5 shows (A) a schematic of the promoter region (-900 to +170) of the human FOXP3 gene and transcription factor binding sites; and (B) a schematic of four HLA-A2 CAR constructs (pTX319, pTX320, pTX321, and pTX322), each under the control of a different FOXP3- derived promoter (hFXP3.1, hFXP3.2, hFXP3.3, and hFXP3.4, respectively). TSS: transcription start site. Each CAR construct comprises the following coding sequences: CD8 signal peptide, an ScFv directed against HLA-A2, human CD8 linker and transmembrane domain, intracellular human CD28 co-stimulatory domain, and 093z signaling domain.

FIG. 6 is a panel of graphs showing the expression of four HLA-A2 CAR constructs in Tregs cultured with different concentrations (50, 200, or 1,000 U/ml) of interleukin-2 (IL-2). Treg cells were transduced with recombinant lentiviruses carrying the above four HLA-A2 CAR constructs (TX319, TX320, TX321, and TX322, under minimal promoters hFXP3.1, hFXP3.2, hFXP3.3, and hFXP3.4, respectively). Experiment 1 (left graphs) used a high viral titer for transduction (5 X 10⁶ TU/ml), while Experiment 2 (right graphs) used a low viral titer (2 X 10⁶ TU/ml). An HLA-A2 CAR construct under the control of a PGK promoter (TX300) was used as a positive control. The transduction efficiency (indicated by % cells stained positive for HLA-A2 dextramer) and CAR density (as indicated by mean fluorescent intensity or MFI) were assessed at day 7 post transduction. Treg cells were cultured in the presence of 50, 200 or 1,000 U/ml of IL-2 (left to right for each construct on the graphs). The indicated fold decrease in CAR expression is relative to cells transduced with TX300 and cultured in the presence of 1000 U IL-2. NT: not transduced.

FIG. 7 is a panel of graphs showing HLA-A2 CAR density overtime in Treg cells cultured with different concentrations of IL-2 (50 U, 200 U, or 1,000 U/ml). Treg cells were transduced with recombinant lentiviruses carrying HLA-A2 CAR constructs TX300, TX319, TX320, TX321 and TX322. CAR density (MFI) was evaluated at days 3 (D3), 5 (D5) and 7 (D7) using HLA-A2 dextramer staining. NT: not transduced.

FIG. 8 is a graph showing FOXP3 expression in Treg cells. Five days post-transduction, FOXP3 expression was evaluated following intracellular staining in un-transduced (UT) or TX319-transduced Treg cells cultured in 50, 200 or 1,000 U/ml of IL-2 (left to right for each group of three bars).

FIG. 9 is a set of dot plots showing transduction efficiency and CD20 CAR expression at the Treg cell surface. Transduction efficiency was assessed at Day 7 using GFP expression levels and CAR density (% and MFI) was assessed using protein-L labeling.

FIG. 10 is a set of graphs evaluating ligand-independent tonic signaling and activation capacity of the CD20 CAR. Transduced FOXP3⁺ Tregs were seeded alone (Panels A, B), in the presence of anti-CD3/anti-CD28 coated beads (Panel B), or in the presence of freshly thawed autologous B cells (Panel B). After 24 hours, the cells were stained for CD4 and CD69 cell surface expression.

FIG. 11 is a set of graphs showing that CD20 CAR at a low level of expression exhibit efficient CAR-mediated suppressive activity. Contact-dependent suppression mediated by CAR Treg cells in the absence of any activation (“None”), after TCR activation (“CD3/CD28 beads”) or after B cell-induced CAR activation (“B cells”) was evaluated by measuring the proliferation of conventional T cells (Tconv).

FIG. 12 is a panel of schematic and graphs. (A) shows a schematic of the following four IL23R CAR constructs: Constructs TX418 and TX417 comprise the hPGK promoter, while constructs TX420 and TX419 comprise minimal promoter hFXP3.1. Constructs TX418 and TX420 further comprise the WpreMut6 regulatory region. (B) shows a panel of graphs showing the quantification of cell surface expression of these four IL23R CAR constructs in effector T cells (Jurka-Lucia NFAT cells). T cells were transduced with recombinant lentiviruses carrying the above four IL23R CAR constructs (TX418 TX417, TX420, and TX419).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides an expression construct for a protein of interest that is not a human FOXP3 protein, wherein expression of the protein is driven by a promoter derived from a human FOXP3 gene. The promoter comprises a core, functional region of the natural human FOXP3 promoter and is especially suitable for expressing a protein such as a CAR, a CAAR or a TCR of interest in a T cell (e.g., a T effector cell or a Treg cell) at a level that is sufficient for the protein to achieve its biological function, yet low enough to avoid causing exhaustion of the T cell or ligand-independent tonic signaling. T cell exhaustion is characterized by progressive loss of T cell functions, and it prevents the persistence of the engineered T cells in a patient. T cell exhaustion has been an obstacle encountered in CAR T therapy. The present expression constructs are expected to provide suitable expression levels of the protein to allow the genetically modified T cell (e.g., the genetically modified CAR T cell) to persist in a patient.

The present FOXP3- derived promoters (e.g., minimal or truncated FOXP3 promoters) can be used to drive the expression of CAR in T cells such as regulatory T cells. Regulatory T cells maintain immune homeostasis and confer immune tolerance. Engineered Treg cells comprising the present expression constructs can be used in cell-based therapy to treat patients in need of induction of immune tolerance or restoration of immune homeostasis, such as patients receiving organ transplantation or allogeneic cell therapy (e.g., patients with an organ transplantation condition) and patients with an autoimmune disease. Engineered Treg cells comprising the present expression constructs can also be used in cell-based therapy to treat patients with an inflammatory disease. Engineered Treg cells comprising the present expression constructs can also be used in cell-based therapy to treat patients with an allergic disease. The present Treg cells may have enhanced immune-regulatory activities, including improved tissue-specificity (e.g., through expression of a receptor specific for an antigen in a targeted tissue). The Tregs will actively control the proliferation and activation of T effector cells locally and/or systemically through receptor-mediated cell-to-cell contact and/or secretion of immunosuppressive cytokines (e.g., IL-10, TGF-b and IL-35).

Since the engrafted Tregs can proliferate and self-renew, the present cell therapy can achieve long-term tolerance and protection of the transplant. See, e.g., Dawson et al., JCI Insight. (2019) 4(6):el23672, which is incorporated herein by reference in its entirety.

I. Expression Constructs Containing FOXP3- Derived Promoters The expression constructs of the present disclosure comprise a promoter derived from a human FOXP3 gene. The term “derived from” as used herein, indicates a relationship between a first molecule and a second molecule. It generally refers to structural similarity between the two molecules and does not require that one of them be physically generated from the other one. In the present invention, a promoter “derived from” a human FOXP3 gene refers to a promoter that contains a functionally active region of the promoter of a human FOX3 gene, or a functional equivalent thereof (e.g., a variant of the active region containing nucleotide changes from the native sequence where the nucleotide changes do not adversely affect the promoter’s Treg specificity or the promoter’s transcription regulatory activity). The nucleotide sequence of a human FOXP3 gene is available at NCBI, Reference Sequence No. NC_000023.11 ( Homo sapiens chromosome X, GRCh38.pl3 Primary Assembly). The promoter region of the gene encompasses at least several kilobases. The present inventors have unexpectedly discovered that a much smaller portion of this region contains the core, or minimally required, transcriptionally active sequence of the promoter. The inventors have also discovered that truncated FOXP3 promoters containing this core have a weak, yet effective, activity to drive expression of a CAR construct at a level that does not exhaust T cells. This core portion resides within nucleotide 49266500 to nucleotide 49264696 of NC_000023.11, corresponding to nucleotide -1,799 to nucleotide 1 (i.e., transcription start site) of the gene.

The FOXP3- derived promoters exemplified herein include, without limitations:

(l) hFXP3.1 (903 bp): -900 to +3 (Bases 49265601 to 49264699 of the GRCh38.pl2 Primary Assembly) (SEQ ID NO: 1);

(n) hFXP3.2 (1072 bp): -900 to +172 (Bases 49265601 to 49264530 of the Primary Assembly) (SEQ ID NO: 2);

(ill) hFXP3.3 (1802 bp): -1799 to +3 (Bases 49266500 to 49264699 of the Primary Assembly) (SEQ ID NO: 3); and

(IV) hFXP3.4 (1971 bp): -1799 to +172 (Bases 49266500 to 49264530 of the Primary Assembly) (SEQ ID NO: 4).

In some embodiments, the promoter includes the Stat5 binding domains in the native FOXP3 promoter, e.g., including nucleotides -769 to -729 (SEQ ID NO: 5) of the human FOXP3 gene (FIG. 5A).

In some embodiments, the promoter includes additional sequences downstream of the Stat5 binding domains, e.g., including nucleotides -729 to 0, and/or additional sequences upstream of the Stat5 binding domains, e.g., including nucleotides -1,799 to -769 or nucleotides -900 to -769. In some embodiments, the promoter includes nucleotides -900 to 0, or -1,799 to 0. In some embodiments, the promoter may also contain FOXP3 gene sequences downstream of the transcription start site (nucleotide 0), e.g., including nucleotides 0 to 3, or nucleotides 0 to +200, or nucleotides 0 to nucleotides +172. Thus, the promoter of the present expression construct may include a sequence (e.g., more than 100, 200, 400, 600, 800, 1,000, 1,200, 1,400, 1,600, or 1,800 bp) within nucleotides -1,800 to +200 of the human FOXP3 gene. Due to the vector size limitation, in certain embodiments, the promoter does not exceed about 2 kb (e.g., does not exceed 2.2 kb) in size so as to allow the inclusion of a large protein-encoding sequence on the vector.

As used herein, a human FOXP3 gene refers to a FOXP3 gene from any human individual. It may contain genetic variations from the specific sequences shown here. The present disclosure contemplates all functional equivalents of the specific promoter sequences shown here. By “nucleotides X to Y” of a human FOXP3 gene is meant nucleotides X to Y of the human FOXP3 exemplified at NCBI, Sequence Reference No. NC_000023.11, or a functional equivalent thereof (including, for example, a sequence containing minor nucleotide changes, and a sequence from an equivalent genomic region in a different human FOXP3 allele).

The coding sequence on the present expression construct may encode a protein of interest such as those encoding a TCR, a CAAR, or a CAR such that the Tregs expressing the TCR or CAR are specific for an antigen of interest.

A CAAR is a chimeric receptor comprising an extracellular domain derived from an autoantigen (e.g., an autoantigen involved in an autoimmune disease), a transmembrane domain, and an intracellular signaling domains. In some embodiments, a CAAR further comprises an extracellular hinge domain, a tag, and/or a leader sequence. The intracellular signaling domain generates a signal that promotes an immune effector function of the cell expressing the CAAR. Examples of immune effector functions may include cytolytic activity, suppressive activity, regulatory activity, and helper activity, including the secretion of cytokines.

A CAR is a fusion protein designed to target T cells expressing it to a desired antigen. In its most basic form, a CAR comprises an extracellular antigen-binding domain and a series of customized intracellular TCR costimulatory/signaling domains. Once the CAR binds to its antigen, it induces similar activation of the cell expressing it, as a natural TCR would. Antigen-specific engineered Tregs enable enhanced immune suppression by homing to the targeted tissue (e.g., a transplant or a site of autoimmune inflammation or allergic reaction). They can interact with Teff cells that are specific, for example, for an allo-antigen (in cases of transplantation or of allergic diseases, for example) or an autoantigen (in cases of autoimmune disease for example). CARs offer the advantage that, unlike natural TCRs, they bind to antigens without the need to interact with other co-stimulatory molecules or involvement of MHC class I or II molecules, thereby affording them functionality in broader settings. In some embodiments, a CAR comprises an extracellular antigen-binding domain, optionally an extracellular hinge domain, a transmembrane domain, an intracellular signaling domain and optionally a tag and/or leader sequence.

A. Antigen-Binding Domains of CARs

The antigen-binding domain of a CAR may comprise an antibody fragment such as an scFv, a Fv, a Fab, a (Fab’)2, a single domain antibody (SDAB), a VH or VC domain, or a camelid VFIH domain.

In some embodiments, the CAR is specific for a polymorphic allogeneic MHC molecule, such as one expressed by cells in a solid organ transplant or by cells in a cell-based therapy (e.g., bone marrow transplant, cancer CAR T therapy, or cell-based regenerative therapy). MHC molecules so targeted include, without limitation, HLA-A, HLA-B, or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR. By way of example, the CAR targets class I molecule HLA-A2. HLA-A2 is a commonly mismatched histocompatibility antigen in transplantation. HLA-A mismatching is associated with poor outcomes after transplantation. Engineered Tregs expressing a CAR specific for an MHC class I molecule are advantageous because MHC class I molecules are broadly expressed on all tissues, so the Tregs can be used for organ transplantation regardless of the tissue type of the transplant. CAR against HLA-A2 offers the additional advantage that HLA-A2 is expressed by a substantial proportion of the human population and therefore on many donor organs. Non-limiting examples of HLA-A2 CAR are those having the amino acid sequence of SEQ ID NO: 10, 20, or 23; CARs having the VH and/or VL sequences of those exemplified CARs; and functional variants of those CARs, such as those having the CDRs of the exemplified VH and VL but different framework sequences. See, e.g., WO 2018/183293 and WO 2019/056099. There has been evidence showing that expression of an HLA-A2 CAR in Treg cells can enhance the potency of the Treg cells in preventing transplant rejection (see, e.g., Boardman, supra,· MacDonald et al, J Clin Invest. (2016) 126(4): 1413-24; and Dawson, supra). In some embodiments, the CAR is specific for an autoantigen, i.e., an endogenous antigen expressed prevalently or uniquely at the site of autoimmune inflammation in a specific tissue of the body. Tregs expressing such a CAR can home to the inflamed tissue and exert tissue-specific activity by causing local immunosuppression. Examples of autoantigens are aquaporin water channels (e.g., aquaporin-4 water channel), paraneoplastic antigen Ma2, amphiphysin, voltage-gated potassium channel, N-methyl-d-aspartate receptor (NMDAR), a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR), thyroid peroxidase, thyroglobulin, anti-N-methyl-D-aspartate receptor (NR1 subunit), Rh blood group antigens, desmoglein 1 or 3 (Dsgl/3), BP180, BP230, acetylcholine nicotinic postsynaptic receptors, thyrotropin receptors, platelet integrin, glycoprotein Ilb/IIIa, calpastatin, citrullinated proteins, alpha-beta-crystallin, intrinsic factor of gastric parietal cells, phospholipase A2 receptor 1 (PLA2R1), and thrombospondin type 1 domain-containing 7A (THSD7A). Additional examples of autoantigens are multiple sclerosis-associated antigens (e.g., myelin basic protein (MBP), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), oligodendrocyte myelin oligoprotein (OMGP), myelin associated oligodendrocyte basic protein (MOBP), oligodendrocyte specific protein (OSP/Claudin 11), oligodendrocyte specific proteins (OSP), myelin-associated neurite outgrowth inhibitor NOGO A, glycoprotein Po, peripheral myelin protein 22 (PMP22), 2’3 ’-cyclic nucleotide 3 ’-phosphodiesterase (CNPase), and fragments thereof); joint-associated antigens (e.g., citrulline-substituted cyclic and linear filaggrin peptides, type II collagen peptides, human cartilage glycoprotein 39 peptides, keratin, vimentin, fibrinogen, and type I, III, IV, and V collagen peptides); and eye-associated antigens (e.g., retinal arrestin, S-arrestin, interphotoreceptor retinoid-binding proteins, beta-crystallin Bl, retinal proteins, choroid proteins, and fragments thereof).

In some embodiments, the antigen targeted by the CAR or TCR expressed by the cell of the invention (in particular by the Treg cells of the invention) is IL23-R (for treatment of, e.g., Crohn’s disease, inflammatory bowel disease, or rheumatoid arthritis), MOG (for treatment of multiple sclerosis), MBP (for treatment of multiple sclerosis), ovalbumin (for treatment of inflammatory bowel disease) or type II collagen (for treatment of an arthritic condition or of uveitis).

In some embodiments, the TCR or CAR may target other antigens of interest (e.g., B cell markers CD19 and CD20 (e.g., SEQ ID NO: 24)). Additionally, rather than using two separate CAR constructs, the CAR transgene itself may encode a bi-specific CAR capable of recognizing, e.g., both CD19 and CD20 (Zah et al., Cancer Immunol Res. (2016) 4(6):498-508).

In some embodiments, the CAR is specific for cancer antigen. As used herein, the term “cancer antigen” refers to an antigen that is differentially expressed by cancer cells and can therefore be exploited to target cancer cells. Cancer antigens are antigens that can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells; these antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Many tumor antigens have been defined in terms of multiple solid tumors: MAGE 1, 2, & 3 (defined by immunity), MART-l/Melan-A, gplOO, carcinoembryonic antigen (CEA), HER2, mucins (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, viral proteins such as some encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

Other cancer antigens include, but are not limited to, 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin/m, b-catenin/mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region- Abelson), CAIX (carbonic anhydrase IX), CD 19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exons 7/8),

CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide- 1 ), CASP-8 (caspase-8), CDC27m (cell-division cycle 27 mutated), CDK4/m (cycline-dependent kinase 4 mutated), CEA (carcinoembryonic antigen),

CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialoganglioside 2),

GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V),

GplOO (glycoprotein lOOkD), HAGE (helicose antigen), HER-2/neu (human epidermal receptor-2/neurological; also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (human papilloma virus), HSP70- 2M (heat shock protein 70-2 mutated), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase), IL-13R-a2 (Interleukin- 13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), k-light chain, LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase), LeY (Lewis-Y antibody), LI CAM (LI cell adhesion molecule), MAGE (melanoma antigen), MAGE-Al (melanoma-associated antigen 1 ), mesothelin, murine CMV infected cells, MART-l/Melan-A (melanoma antigen recognized by T cells- I/melanoma antigen A), MCI R (melanocortin 1 receptor), yosin/m (myosin mutated), MUC1 (mucin 1 ), MUM-1 , -2,-3 (melanoma ubiquitous mutated- 1, -2, -3), NA88-A (NA cDNA clone of patient M88), NKG2D (natural killer group 2, member D) ligands, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), oncofetal antigen (h5T4), PI 5 (protein 15), pi 90 minor bcr-abl (protein of 190KD bcr-abl), Pml/RARa (promyelocytic leukaemia/retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3), synovial sarcoma X-l, -2, -3, -4 (SSX-1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2 (TRP-2/intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms’ tumor gene).

In some embodiments, the TCR or CAR is multispecific, and comprises a fragment of a bispecific antibody. Consequently, in some embodiments, the TCR or CAR is able to bind to two different antigens, or to two different epitopes on the same antigen.

B. Hinge Domains

In some embodiments, the extracellular binding domain is connected to a transmembrane domain by a spacer domain or a hinge domain. Examples of linkers include, but are not limited to, GS linkers as described herein. In certain embodiments, the linker may comprise or consist of the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 32).

In some embodiments, a short oligo- or polypeptide linker, having a length ranging from, e.g., 2 and 10 amino acids, may form the hinge domain. In some embodiments, the term “linker” refers to a flexible polypeptide linker. For example, a glycine-serine doublet may provide a suitable hinge domain (GS linker). In some embodiments, the hinge domain is a Gly/Ser linker. Examples of Gly/Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, and GiS linkers.

Examples of G2S linkers include, but are not limited to, GGS.

G3S linkers comprise the amino acid sequence (Gly-Gly-Gly-Ser)n, also referred to as (GGGS)n or (SEQ ID NO: 33)_n, where n is a positive integer equal to or greater than 1 (such as, for example, n=l, n=2, n=3. n=4, n=5, n=6, n=7, n=8, n=9 or n=10). Examples of G3S linkers include, but are not limited to, GGGSGGGSGGGSGGGS (SEQ ID NO: 34).

Examples of GiS linkers include, but are not limited to, (Gly4 Ser) corresponding to GGGGS (SEQ ID NO: 35); (Gly4 Ser)2 corresponding to

GGGGSGGGGS (SEQ ID NO: 36); (Gly₄Ser)₃ corresponding to GGGGS GGGGS GGGGS (SEQ ID NO: 13); and (Gly₄ Ser) corresponding to GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 37).

In some embodiments, a spacer domain may have a length of up to 300 amino acids, e.g., 10-100 amino acids, 25-50 amino acids, or 2-10 amino acids.

In some embodiments, the hinge domain is a short oligo- or polypeptide linker, e.g., having a length ranging from 2 to 10 amino acids, as described herein. An example of a hinge domain that may be used in the present invention is described in PCT Patent Publication WO2012/138475, incorporated herein by reference.

In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of the amino acid sequence AGSSSSGGSTTGGSTT (SEQ ID NO: 38), the ammo acid sequence

GTTAASGSSGGSSSGA (SEQ ID NO: 39), the amino acid sequence

SSATATAGTGSSTGST (SEQ ID NO: 40), and the amino acid sequence TSGSTGTAASSTSTST (SEQ ID NO: 41). In some embodiments, the hinge domain is encoded by a nucleotide sequence of GGTGGCGGAGGTT CT GGAGGT GGAGGTT CC (SEQ ID NO: 42).

In some embodiments, the hinge domain is a KIR2DS2 hinge corresponding to KIRRDSS (SEQ ID NO: 43).

In some embodiments, the hinge domain comprises or consists of the amino acid sequence of a CD8 hinge (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 15) or an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15. In some embodiments, the hinge domain is a CD8 hinge encoded by the nucleic acid sequence of SEQ ID NO: 44 or a nucleic acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44.

In some embodiments, the hinge domain comprises or consists of the amino acid sequence of a IgG4 hinge (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 45), or an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45. In some embodiments, the hinge domain is an IgG4 hinge encoded by the nucleic acid sequence of SEQ ID NO: 46 or a nucleic acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 46.

In some embodiments, the hinge domain comprises or consists of the amino acid sequence of an IgD hinge (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 47) or an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 47. In some embodiments, the hinge domain is an IgD hinge encoded by the nucleic acid sequence of SEQ ID NO: 48 or a nucleic acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 48. In some embodiments, the hinge region comprises or consists of the amino acid sequence of a CD28 hinge (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 49) or an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 49. In some embodiments, the hinge domain is a CD28 hinge encoded by the nucleic acid of SEQ ID NO: 50 or a nucleic acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 50.

The term “homology” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. Thus, the term “homologous” or “identical,” when used in a relationship between the sequences of two or more polypeptides or of two or more nucleic acid molecules, refers to the degree of sequence relatedness between polypeptides or nucleic acid molecules, as determined by the number of matches between strings of two or more amino acid or nucleotide residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G, Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48:1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Exemplary computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al, Nucl. Acid. Res. 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al, J. Mol. Biol. 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.

C. Transmembrane Domains

Examples of transmembrane domains that may be used in a CAR or CAAR of the invention include, but are not limited to, transmembrane domains of TNFR2, CD28, CD8, or of an alpha, beta or zeta chain of a T cell receptor, or of CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD 9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4- IBB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CD lib, PD1, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. In some embodiments, the transmembrane domain may comprise the entire transmembrane domain of the molecule from which it is derived, or it may comprise a functional fragment or variant thereof.

D. CAR Co-Stimulatory and Stimulatory/Activating Domains

The CAR or CAAR may comprise one or more transmembrane domains from one or more immune cell surface molecules. In some embodiments, the intracellular signaling domain of the CAR or CAAR comprises one or more intracellular costimulatory and activating domains from one or more immune cell surface molecules. In some embodiments, the activating domain comprises or consists of one or more T cell primary signaling domains (or a sequence derived therefrom).

In some embodiments, a CAR or CAAR may comprise the transmembrane and/or intracellular portion of a costimulatory molecule on a T cell. A costimulatory molecule on a T cell binds to its ligand on an antigen-presenting cell in concert with the TCR’s binding to the antigen on the antigen-presenting cell, and allows the activation (e.g., proliferation and secretion of cytokines) of the antigen-bound T cell. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Domains useful in constructing CARs or CAARs may include, without limitation, transmembrane and/or intracellular domains from CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8P, CD28, CD137 (4-1BB), TNFR2, and inducible T cell co-stimulator (ICOS).

In some embodiments, the intracellular domain of a CAR or CAAR of the invention comprises one or more T cell primary signaling domains (or sequence(s) derived therefrom) and optionally one or more intracellular domain(s) of a T cell costimulatory molecule (or sequence(s) derived therefrom). In some embodiments, the intracellular domain may comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or variant thereof. In some embodiments, the intracellular signaling domain consists of at least one primary signaling domain (e.g., a T cell primary signaling domain) or a fragment or variant thereof.

In some embodiments, the intracellular signaling domain consists of at least one costimulatory signaling domain (e.g., a T cell costimulatory molecule intracellular domain) or a fragment or variant thereof.

In some embodiments, the intracellular signaling domain comprises one or more intracellular domain(s) of a T cell costimulatory molecule or a fragment or variant thereof. In some embodiments, the intracellular signaling domain consists of one or more intracellular domain(s) of a T cell costimulatory molecule or a fragment or variant thereof.

In another embodiment, the intracellular signaling domain comprises at least one costimulatory domain or a fragment or variant thereof and at least one primary signaling domain or a fragment or variant thereof.

In another embodiment, the intracellular signaling domain consists of one costimulatory domain or a fragment or variant thereof and one primary signaling domain or a fragment or variant thereof.

In some embodiments, the intracellular signaling domain comprises at least one, two, three, or four costimulatory domains or a fragment or variant thereof and at least one primary signaling domain or a fragment or variant thereof. In certain embodiments, one or more of the costimulatory domains are intracellular domains of a T cell costimulatory molecule. In certain embodiments, the at least one primary signaling domain is a T cell primary signaling domain.

Examples of intracellular domains of a T cell costimulatory molecule include, but are not limited to, the signaling domains of proteins selected from the group consisting of TNFR2 (CD 120b/TNFRSF IB), 4-1BB (CD137), ICOS (CD278), CD27, CD28, CTLA-4 (CD152), PD-1, an MHC class I molecule, BTLA, a Toll ligand receptor, 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, FIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, ARHR, BALER, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl ), NKp44, NKp30, NKp46, CD 160 (BY55), CD 19, CD 19a, CD4, CD8alpha, CD8beta, IL2ra, IL6Ra, IL2R beta, IL2R gamma, IL7R alpha, IL-13RA1/RA2, IL-33R(IL1RL1), IL-10RA/RB, IL-4R, IL-5R (CSF2RB), IL-21R, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CD 11 a/CD 18, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, CD95, TNFR1 (CD 120a/TNFRSF 1 A), TGFbRl/2/3, TRAN CE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, common gamma chain, a ligand that specifically binds with CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof. See also, e.g., Chen and Flies, Nat Rev Immunol. (2013) 13(4):227-42.

The activating domain or the T cell primary signaling domain of the CAR may be derived from CD3-zeta or CD3 -epsilon. The CD3-zeta chain may have the protein sequence provided as GenBank Ace. No. BAG36664.1, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape and the like). A CD3-zeta activating or stimulatory domain includes the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Ace. No. BAG36664.1 or the equivalent residues from a non-human species.

In some embodiments, the activating domain or the T cell primary signaling domain comprises a signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAPIO, and DAP12, and sequences derived therefrom. In some embodiments, the T cell primary signaling domain that comprises or consists of at least one functional signaling domain of CD3 zeta or a fragment or variant thereof.

In some embodiments, the protein expressed by the present expression construct may include an epitope tag to allow monitoring of the gene integration and expression. Said tag may be localized N-terminally, C-terminally and/or internally. Epitope tags include, for example, one or more copies of FLAG (e.g., 3x flag tag), His tag, myc tag, Tap tag, HA tag, low-affinity nerve growth factor receptor (LNGFR) and/or its antibody-binding domain as a tag, or any other readily detectable amino acid sequence. Other examples of tags include, without limitation, a tag selected from the group consisting of streptavidin tag (e.g., of SEQ ID NO: 28), hemagglutinin tag, poly arginine tag, S-tag, HAT tag, calmodulin-binding peptide tag, SBP tag, chitin binding domain tag, GST tag, maltose-binding protein tag, fluorescent protein tag, T7 tag, V5 tag and Xpress tag. Other examples of tags include, without limitation, NWSHPQFEK (SEQ ID NO: 51) or SAWSHPQFEK (SEQ ID NO: 52).

In some embodiments, a CAR of the invention further comprises P2A (SEQ ID NO: 30) and/or GFP (SEQ ID NO: 31) sequences.

In some embodiments, the coding sequence on the construct may also encode a signal or leader peptide to facilitate the surface expression of the transgene. For example, the signal sequence may be one derived from the signal sequence of human GM-CSF or CD8.

Additional elements may be included on the construct. For example, to allow transcription termination, the construct may include a polyadenylation (polyA) site such as an SV40 polyA site. The heterologous sequence may also include RNA-stabilizing elements such as a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (Wpre) or a derivative thereof such as WpreMut6.

In some embodiments, the construct is a vector selected from the group comprising or consisting of DNA vectors, RNA vectors, cosmids, plasmids (e.g., naked or contained in liposomes), phagemids, phage derivatives, transposons ( e.g ., sleeping beauty) or viral vectors (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

II. Engineered Cells Containing the Expression Constructs

The present invention further relates to engineered cells or to a population of engineered cells comprising one or more constructs as described herein. In one embodiment, the construct is integrated into the genome of the engineered cells. In one embodiment, the engineered cells are engineered immune cells. In some embodiments, the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, ab T cells, gd T cells, double negative (DN) cells, regulatory immune cells, regulatory T (Treg) cells, effector immune cells, effector T cells (such as, for example, CD4⁺ and CD8⁺ effector T cells), B cells, and myeloid-derived cells, and any combination thereof, wherein the immune cells are optionally human cells.

The present invention further relates to a composition, to a pharmaceutical composition and to a medicament comprising at least one genetically engineered cell as described herein, or at least one population thereof.

The engineered cells of the present disclosure may thus be provided in a pharmaceutical composition containing the cells and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition comprises sterilized water, physiological saline or neutral buffered saline (e.g., phosphate-buffered saline), salts, antibiotics, isotonic agents, and other excipients (e.g., glucose, mannose, sucrose, dextrans, mannitol; proteins (e.g., human serum albumin); amino acids (e.g., glycine and arginine); antioxidants (e.g., glutathione); chelating agents (e.g., EDTA); and preservatives). The pharmaceutical composition may additionally comprise factors that are supportive of the Treg phenotype and growth (e.g., IL-2 and rapamycin or derivatives thereof), anti-inflammatory cytokines (e.g., IL-10, TGF-b, and IL-35), and other cells for cell therapy (e.g., CAR T effector cells for cancer therapy or cells for regenerative therapy). For storage and transportation, the cells optionally may be cryopreserved. Prior to use, the cells may be thawed and diluted in a pharmaceutically acceptable carrier.

The engineered cells of the present disclosure are mammalian cells, such as human cells, cells from a farm animal (e.g., a cow, a pig, or a horse), and cells from a pet (e.g., a cat or a dog). The expression constructs described herein may be introduced to Treg cells, or on cells that are not Treg cells but are differentiated into Treg cells after the introduction of the expression construct. The Treg phenotype is in part dependent on the expression of the master transcription factor forkhead box P3 (FOXP3), which regulates the expression of a network of genes essential for immune suppressive functions.

As used herein, the terms “regulatory T cells,” “regulatory T lymphocytes,” and Tregs refers to a subpopulation of T cells that modulates the immune system, maintains tolerance to self-antigens, and generally suppresses or downregulates induction and proliferation of T effector cells. Tregs often are marked by the phenotype of CD4⁺CD25⁺CD127^loFOXP3⁺. In some embodiments, Tregs are also CD45RA⁺, CD62L¹", and/or GITR⁺. In particular embodiments, Tregs are marked by CD4⁺CD25⁺CD127^loCD62L⁺ or €ϋ4Aΐϊ)45KAAΐϊ)25^M€0127¹⁰. As used herein, Tregs include (i) “natural” Tregs that develop in the thymus; (ii) induced, adaptive, or peripheral Tregs arising via a differentiation process that takes place outside the thymus (e.g., in tissues or secondary lymphoid organs, or in laboratory settings under defined culture conditions); and (iii) Tregs that have been created using recombinant DNA technology, including genome editing and gene therapy.

In some embodiments, the constructs of the present disclosure are introduced to T effector cells.

A. Isolation of Treg cells

The Treg cells may be isolated from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, or spleen tissue. For example, Tregs may be isolated from a unit of blood collected from a subject using well known techniques such as Ficoll™ separation, centrifugation through a PERCOLL™ gradient following red blood cell lysis and monocyte depletion, counterflow centrifugal elutriation, leukapheresis, and subsequent cell surface marker-based magnetic or flow cytometric isolation.

Further enrichment of Treg cells from the isolated white blood cells can be accomplished by positive and/or negative selection with a combination of antibodies directed to unique surface markers using techniques such as flow cytometry cell sorting and/or magnetic immunoadherence involving conjugated beads. For example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically may include antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8. To enrich or positively select for Tregs, antibodies to CD4, CD25, CD45RA, CD62L, GITR, and/or CD127 can be used.

In an exemplary and nonlimiting protocol, Treg cells may be obtained as follows ( see Dawson et al., JCI Insight. (2019) 4(6):el23672). CD4⁺ T cells are isolated from a human donor via RosetteSep (STEMCELL Technologies, 15062) and enriched for CD25⁺ cells (Miltenyi Biotec, 130-092-983) prior to sorting live CD4⁺CD25^hiCD127¹° Tregs or CD4⁺CD127^loCD25^hiCD45RA⁺ Tregs using a MoFlo Astrios (Beckman Coulter) or FACSAria II (BD Biosciences). Sorted Tregs may be stimulated with L cells and anti-CD3 monoclonal antibody (e.g., OKT3, UBC AbLab; 100 ng/ml) in ImmunoCult-XF T cell expansion media (STEMCELL Technologies, 10981) with 1000 U/ml IL-2 (Proleukin) as described in MacDonald et al., J Clin Invest. (2016) 126(4): 1413-24). One or more days later, the Treg cells may be genetically modified as described below. For phenotypic analysis, cells may be stained with fixable viability dye (FVD, Thermo Fisher Scientific, 65-0865-14; BioLegend, 423102) and for surface markers before fixation and permeabilization using an eBioscience FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, 00-5523-00) and staining for intracellular proteins. Samples may be read on a CytoFLEX (Beckman Coulter). Tregs may also be derived from T effector cells in vitro , for example, by exposure to IL-10 or TGF-b.

Plasticity is a property inherent to nearly all types of immune cells. It appears that Treg cells are able to transition (“drift”) to Teff cells under inflammatory and environmental conditions (Sadlon et al., Clin Transl Imm. (2018) 7(2):el011). To maintain the Treg phenotype and/or to increase expression of FOXP3 and the transgene in the engineered Treg cells, the cells may be cultured in tissue culture media containing rapamycin and/or a high concentration of IL-2. See, e.g., MacDonald et al., Clin Exp Immunol. (2019) doi: 10.1111/cei.13297.

B. Isolation of Non-Treg Cells

The source cells, i.e., cells into which the expression construct is introduced, may also be pluripotent stem cells (PSCs). PSCs are cells capable of giving rise to any cell type in the body and include, for example, embryonic stem cells (ESCs), PSCs derived by somatic cell nuclear transfer, and induced PSCs (iPSCs). See, e.g., Iriguchi and Kaneko, Cancer Sci. (2019) 110(1): 16-22 for differentiating iPSCs to T cells. As used herein, the term “embryonic stem cells” refers to pluripotent stem cells obtained from early embryos; in some embodiments, this term refers to ESCs obtained from a previously established embryonic stem cell line and excludes stem cells obtained by recent destruction of a human embryo.

In other embodiments, the source cells for genome editing are multipotent cells such as hematopoietic stem cell (HSCs such as those isolated from bone marrow or cord blood), hematopoietic progenitor cells (e.g., lymphoid progenitor cell), or mesenchymal stem cells (MSC). Multipotent cells are capable of developing into more than one cell type, but are more limited than cell type potential than pluripotent cells. The multipotent cells may be derived from established cell lines or isolated from human bone marrow or umbilical cords. By way of example, the HSCs may be isolated from a patient or a healthy donor following G-CSF-induced mobilization, plerixafor- induced mobilization, or a combination thereof. To isolate HSCs from the blood or bone marrow, the cells in the blood or bone marrow may be panned by antibodies that bind unwanted cells, such as antibodies to CD4 and CD8 (T cells), CD45 (B cells), GR-1 (granulocytes), and lad (differentiated antigen- presenting cells) (see, e,g., Inaba, el al. (1992) J. Exp. Med. 176:1693-1702). HSCs can then be positively selected by antibodies to CD34.

In still other embodiments, the source cells are non-Treg lymphoid cells that are differentiated into Treg cells after genome editing. See above for how to differentiate T effector cells into Treg cells.

The engineered non-Treg cells may be differentiated into Treg cells before engrafting into a patient as described above. Alternatively, the engineered non-Treg cells may be induced to differentiate into Treg cells after engrafting to a patient.

C. Introduction of Expression Constructs into Target Cells

The present expression constructs can be introduced to the target cell by any known techniques such as chemical methods (e.g., calcium phosphate transfection and lipofection), non-chemical methods (e.g., electroporation and cell squeezing), particle- based methods (e.g., magnetofection), and viral transduction (e.g., by using viral vectors such as vaccinia vectors, adenoviral vectors, lentiviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, and hybrid viral vectors). In some embodiments, the construct is an AAV viral vector and is introduced to the target human cell by a recombinant AAV virion whose genome comprises the construct, including having the AAV Inverted Terminal Repeat (ITR) sequences on both ends to allow the production of the AAV virion in a production system such as an insect cell/baculovirus production system or a mammalian cell production system). The AAV may be of any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, or AAVrhlO, of a pseudotype such as AAV2/8, AAV2/5, or AAV2/6. The expression cassette on the construct may be integrated to the host genome by any site-specific gene knockin technique. Such techniques include, without limitation, homologous recombination, gene editing techniques based on zinc finger nucleases or nickases (collectively “ZFNs” herein), transcription activator-like effector nucleases or nickases (collectively “TALENs” herein), clustered regularly interspaced short palindromic repeat systems (CRISPR, such as those using Cas9 or cpfl), meganucleases, integrases, recombinases, and transposes. For site-specific gene editing, the editing nuclease typically generates a DNA break (e.g., a single- or double-stranded DNA break) in the targeted genomic sequence such that a donor polynucleotide having homology to the targeted genomic sequence (e.g., the construct described herein) is used as a template for repair of the DNA break, resulting in the introduction of the donor polynucleotide to the genomic site.

Gene editing techniques are well known in the art. See, e.g., U.S. Pats. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,895,308, 8,906,616, 8,932,814,

8,945,839, 8,993,233, 8,999,641, 9,790,490, 10,000,772, 10,113,167, and 10,113,167 for

CRISPR gene editing techniques. See, e.g., U.S. Pats. 8,735,153, 8,771,985, 8,772,008,

8,772,453, 8,921,112, 8,936,936, 8,945,868, 8,956,828, 9,234,187, 9,234,188, 9,238,803,

9,394,545, 9,428,756, 9,567,609, 9,597,357, 9,616,090, 9,717,759, 9,757,420, 9,765,360,

9,834,787, 9,957,526, 10,072,062, 10,081,661, 10,117,899, 10,155,011, and 10,260,062 for

ZFN techniques and its applications in editing T cells and stem cells. The disclosures of the aforementioned patents are incorporated by reference herein in their entirety.

In gene editing techniques, the gene editing complex can be tailored to target specific genomic sites by altering the complex’s DNA binding specificity. For example, in CRISPR technology, the guide RNA sequence can be designed to bind a specific genomic region; and in the ZFN technology, the zinc finger protein domain of the ZFN can be designed to have zinc fingers specific for a specific genomic region, such that the nuclease or nickase domains of the ZFN can cleave the genomic DNA at a site-specific manner. Components of the gene editing complexes may be delivered into the target cells, concurrent with or sequential to the transgene construct, by well known methods such as electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, lipid nanoparticles, immunoliposomes, poly cation or lipid: nucleic acid conjugates, naked DNA or mRNA, and artificial virions. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. In particular embodiments, one or more components of the gene editing complex, including the nuclease or nickase, are delivered as mRNA into the cells to be edited.

D. Additional Genetic Engineering

The present engineered cells may be further genetically engineered to make the cells more effective, more applicable to a larger patient population, and/or safer.

In some embodiments, the present cells may be allogeneic cells to the patient. In such instances, the cells may be engineered to reduce host rejection to these cells (graft rejection) and/or these cells’ potential attack on the host (graft-versus-host disease). By way of example, the cells may be engineered to have a null genotype for one or more of the following: (i) T cell receptor (TCR alpha chain or beta chain); (ii) a polymorphic major histocompatibility complex (MHC) class I or II molecule (e.g., HLA-A, HLA-B, or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR; or P2-microglobulin (B2M)); (iii) a transporter associated with antigen processing (e.g., TAP-1 or TAP-2); (iv) Class II MHC transactivator (CIITA); (v) a minor histocompatibility antigen (MiHA; e.g., HA-1/A2, HA-2, HA-3, HA-8, HB-IH, or HB-IY); and (vi) any combination thereof. The allogeneic engineered cells may also express an invariant HLA or CD47 to protect the engineered cells from host rejection. These further genetic modifications may be performed by the gene editing techniques known in the art and those described herein.

The further- engineered allogeneic cells are particularly useful because they can be used in multiple patients without compatibility issues. The allogeneic cells thus can be called “universal” and can be used “off the shelf.” The use of “universal” cells greatly improves the efficiency and reduces the costs of adopted cell therapy.

The present engineered cells may additionally contain a “safety switch” in their genomes, such that proliferation of the cells can be stopped when their presence in the patient is no longer desired. A safety switch may, for example, be a suicide gene, which upon administration of a pharmaceutical compound to the patient, will be activated or inactivated such that the cells enter apoptosis. A suicide gene may encode an enzyme not found in humans (e.g., a bacterial or viral enzyme) that converts a harmless substance into a toxic metabolite in the human cell. Examples of suicide genes include, without limitation, genes for thymidine kinases, cytosine deaminases, nitroreductases, intracellular antibodies, telomerases, caspases, and DNases. See, e.g., Zarogoulidis et al., J Genet Syndr Gene Ther. doi: 10.4172/2157-7412.1000139 (2013). In some embodiments, the suicide gene may be a thymidine kinase (TK) gene from Herpes Simplex Virus (HSV). A HSV-TK gene can be turned on so as to kill the cell by administration of ganciclovir, valganciclovir, famciclovir, or the like to the patient.

A safety switch may also be an “on” or “accelerator” switch, a gene encoding a small interfering RNA, an shRNA, or an antisense that interferences the expression of a cellular protein critical for cell survival.

The safety switch may utilize any suitable mammalian and other necessary transcription regulatory sequences. The safety switch can be introduced into the cell through random integration or site-specific integration using gene editing techniques described herein or other techniques known in the art. It may be desirable to integrate the safety switch in a genomic safe harbor such that the genetic stability and the clinical safety of the engineered cell are maintained. Examples of safe harbors are the AAVS1 locus; the ROSA26 locus; the CLYBL locus; the gene loci for albumin, CCR5, and CXCR4; and the locus where the endogenous gene is knocked out in the engineered cells (e.g., the T cell receptor alpha or beta chain gene locus, the HLA gene locus, the CIITA locus, or the P2-microglobulin gene locus).

III. Use of the Engineered Tree Cells

The genetically engineered Treg cells of the present disclosure can be used in cell therapy to treat a patient (e.g., a human patient) in need of modulation of an immune response, in particular in induction of immune tolerance or restoration of immune homeostasis. The terms “treating” and “treatment” refer to alleviation or elimination of one or more symptoms of the treated condition, prevention of the occurrence or reoccurrence of the symptoms, reversal or remediation of tissue damage, and/or slowing of disease progression. The present invention thus relates to genetically engineered Treg cells of the present disclosure for use as a drug or as a medicament.

A patient herein may be one having or at risk of having an undesired inflammatory condition such as an autoimmune disease. Examples of autoimmune diseases are Addison’s disease, AIDS, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, dermatitis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch- Schonlein purpura (HSP), juvenile arthritis, juvenile myositis, Kawasaki disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), polymyositis, pulmonary alveolar proteinosis, multiple sclerosis, myasthenia gravis, neuromyelitis optica, PANDAS, psoriasis, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, systemic lupus erythematosus, thrombocytopenic purpura (TTP), Type I diabetes mellitus, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

In some embodiments, the Tregs are engineered to express a CAR targeting an autoantigen associated with an autoimmune disease, such as myelin oligodendrocyte glycoprotein (multiple sclerosis), myelin protein zero (autoimmune peripheral neuropathy), HIV env or gag protein (AIDS), myelin basic protein (multiple sclerosis), CD37 (systemic lupus erythematosus), CD20 (B-cell mediated autoimmune diseases), and IL-23R (inflammatory bowel diseases such as Crohn’s disease or ulcerative colitis).

A patient herein may be one in need of an allogeneic transplant, such as an allogeneic tissue or solid organ transplant or an allogeneic cell therapy. The Tregs of the present disclosure, such as those expressing CARs targeting one or more allogeneic MHC class I or II molecules, may be introduced to the patient, where the Tregs will home to the transplant and suppress allograft rejection elicited by the host immune system and/or graft- versus-host rejection. Patient in need of a tissue or organ transplant or an allogeneic cell therapy include those in need of, for example, kidney transplant, heart transplant, liver transplant, pancreas transplant, intestine transplant, vein transplant, bone marrow transplant, and skin graft; those in need of regenerative cell therapy; those in need of gene therapy (AAV-based gene therapy); and those in need in need of cancer CAR T therapy.

If desired, the patient receiving the engineered Tregs herein (which includes patients receiving engineered pluripotent or multipotent cells that will differentiate into Tregs in vivo) is treated with a mild myeloablative procedure prior to introduction of the cell graft or with a vigorous myeloablative conditioning regimen.

In some embodiments, the genetically-engineered Treg cells of the invention may be used in the treatment of an inflammatory condition in a subject in need thereof.

In some embodiments, the inflammatory condition comprises inflammatory diseases and inflammation linked to an infection or linked to cancer.

In some embodiments, the inflammatory condition comprises inflammatory diseases and inflammation linked to an autoimmune disease.

In some embodiments, the genetically-engineered Treg cells of the invention may be used in the treatment of one or more allergic disease, disorder, symptom, or condition in a subject in need thereof. In certain embodiments, CAR-modified immune cells of the invention may be used to promote immune tolerance in this context. The present invention thus provides a method of treating an allergic disease, disorder, symptom, or condition in a subject in need thereof, wherein said method comprises administering a therapeutically effective amount of at least one genetically-engineered Treg cell or population as described herein. The present invention also provides at least one genetically-engineered Treg cell or population as described herein ( e.g ., in a composition, pharmaceutical composition or medicament as described herein) for use in the treatment of an allergic disease, disorder, symptom, or condition. The present invention also provides at least one genetically-engineered Treg cell population as described herein (e.g., in a composition, pharmaceutical composition or medicament as described herein) for use in the manufacture of a medicament for treating an allergic disease, disorder, symptom, or condition.

Examples of allergic diseases include, but are not limited to, allergic diseases against an inhaled allergen, an ingested allergen or a contact allergen. Other examples of allergic diseases include, but are not limited to, allergic asthma, hypersensitivity lung diseases, food allergy, atopic dermatitis, allergic rhinitis, allergic rhinoconjunctivitis, chronic urticaria, delayed-type hypersensitivity disorders and systematic anaphylaxis.

In some embodiments, the genetically-engineered Treg cells of the invention may be used in the treatment of an organ transplantation condition, such as, for example, graft rejection or graft- versus-host-disease (GvHD).

Another object of the present invention is a pharmaceutical composition as described herein, for use in treating a patient in need of immunosuppression (e.g., wherein the subject has an autoimmune disease, an inflammatory disease, an allergic disease, an organ transplantation condition or has or will receive tissue transplantation). Another object of the present invention is a pharmaceutical composition as described herein, for use in treating cancer or an infectious disease in a patient in need thereof. IV. Use of the Engineered T effector Cells

The genetically engineered T effector cells of the present disclosure can be used in cell therapy to treat a patient (e.g., a human patient) in need of modulation of an immune response, in particular of induction of an immune response. The present invention thus relates to genetically engineered T effector cells of the present disclosure for use as a drug or as a medicament.

In some embodiments, the patient has a cancer. In some embodiments, the patient has an infectious disease.

Another object of the present invention is a pharmaceutical composition as described herein, for use in treating cancer or an infectious disease in a patient in need thereof.

The term “cancer” encompasses solid tumors and/or liquid tumors.

In some embodiments, the infectious disease is a viral infectious disease. As used herein, a “viral infectious disease” may be an infection caused by any virus that causes a disease or pathological condition in the host. In some embodiments, the infectious disease is a bacterial infectious disease. As used to herein, a “bacterial infectious disease” may be an infection caused by any bacteria that causes a disease or pathological condition in the host.

In some embodiments, the infectious disease is a fungal infectious disease. As used to herein, a “fungal infectious disease” may be an infection caused by any fungus that causes a disease or pathological condition in the host.

In some embodiments, the infectious disease is a parasitic infectious disease. As used to herein, a “parasitic infectious disease” may be an infection caused by any protozoa, helminths, or ectoparasites that cause a disease or pathological condition in the host. In some embodiments, the pharmaceutical composition of the present disclosure is (or is to be) administered to a patient in a therapeutically effective amount through systemic administration (e.g., through intravenous injection or infusion) or local injection or infusion to the tissue of interest (e.g., infusion through the hepatic artery, and injection to the brain, heart, or muscle). The term “therapeutically effective amount” refers to the amount of a pharmaceutical composition, or the number of cells, that when administered to the patient, is sufficient to effect the treatment.

In some embodiments, a single dosing unit of the pharmaceutical composition comprises more than 10⁴ cells (e.g., from about 10⁵ to about 10⁶ cells, from about 10⁶ to about 10¹⁰, from about 10⁶ to 10⁷, from about 10⁶ to 10⁸, from about 10⁷ to 10⁸, from about 10⁷ to 10⁹, or from about 10⁸ to 10⁹ cells). In certain embodiments, a single dosing unit of the composition comprises about 10⁶, about 10⁷, about 10⁸, about 10⁹, or about 10¹⁰ or more cells.

The patient may be administered with the pharmaceutical composition once every two days, once every three days, once every four days, once a week, once every two weeks, once every three weeks, once a month or at another frequency as necessary to establish a sufficient population of engineered cells in the patient.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cardiology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

Example 1: CAR Expression Cassettes Using Promoters of Various Strengths

The human elongation factor 1 alpha (EFla) promoter and the human phosphogly cerate kinase (PGK) promoter are widely used for transgene expression in hematopoietic cells and cell therapy. These promoters are considered as “mild promoters,” in contrast to “strong promoters” such as the viral Long Terminal Repeat (LTR) of various viruses (e.g., HIV and SFFV). Furthermore, many transgene constructs include the Woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (Wpre). Wpre improves the stability of a messenger RNA (mRNA) in which the Wpre is inserted. A potentially safer version of Wpre, WpreMut6, was described in Zanta-Boussif et al., Gene Therapy (2009) 16(5):605-19.

This Example describes a study aimed to evaluate the impact of human PGK and EFla promoters and WpreMut6 on the expression level and efficacy of an anti-HLA-A2 CAR construct in human T regulatory cells. For that, four constructs have been designed and produced in lentiviral vectors: PGK-CAR (pTX135 plasmid), PGK-CAR-WpreMut6

(pTX136 plasmid), EFla- CAR (pTX137 plasmid) and EFla-CAR-WpreMut6 (pTX138 plasmid). They are listed below: pTX135: hPGK-HLA.A2 CAR, pTXl 36: hPGK-HLA. A2 CAR-WpreMut6, pTX137: hEFla-HLA.A2 CAR, and pTX138: hEFla-HLA.A2 CAR-WpreMut6.

Next, four lentiviral vectors carrying the CAR expression cassettes were used to transduce Tregs. The expression level of the HLA-A2 CAR transgene, the functionality of the CAR, and the Treg phenotype stability of the transduced cells were evaluated.

MA TERIALS AND METHODS

The materials and reagents used in the study are shown in Table 1 below.

Table 1 Materials and Reagents

Tregs and PBMC Isolation

Primary Treg cells (CD4⁺CD25⁺CD45RA⁺) were sorted from healthy volunteer HLA-A2 negative frozen leukapheresis. Leukopaks were obtained from HemaCare (USA). The table below specifies the main Leukopak characteristics used for these runs. Table 2 Extract from Leukopak Data Used for Testing

CD4⁺CD25⁺CD127^lowCD45RA⁺ nTreg cells were isolated following the procedure described below. For ID 272 cells, after thawing of the leukapheresis, the enrichment steps were: 1) depleting non-CD4⁺ and CD45RO⁺ contaminating cells with the Dynabeads^® Untouched™ Human CD4 T Cells kit (ThermoFisher, #11346D); and 2) positively selecting CD25 ^lg cells from a sample enriched in CD4⁺CD45RA⁺ cells with the EasySep™ Human CD25 Positive Selection Kit (StemCell, #18231). For ID 320-2 cells, after thawing of the leukapheresis, the enrichment steps were: 1) depleting non-CD4⁺ and CD45RO⁺ contaminating cells with the Dynabeads^® Untouched™ Human CD4 T Cells kit (ThermoFisher, #11346D); and 2) positively selecting CD25^high cells from a sample enriched in CD4⁺CD45RA⁺ cells with the IBA FAB 25 STREPTAMER Kit (IB A, #8004-1016). After the enrichment step, cell sorting was performed with the SH800 sorter (SONY) gated on the CD4⁺CD25^highCD127^low-^negCD45⁺ cells.

HLA-A2 negative or positive PBMCs were isolated from healthy volunteers using standard ficoll procedures. The lot used in this study were PBMCs ID251 (HLA-A2⁺) and ID265 (HLA-A2 ). Design of Different CAR Expression Cassettes

Four HLA-A2 CAR expression cassettes were designed with either a human PGK promoter or a human EFla promoter, with or without WpreMut6. The sequence for the human PGK promoter is shown in SEQ ID NO: 6; this 516 bp sequence is available at GeneBank Accession No. NG 008862.1 ( homo sapiens phosphoglycerate kinase 1 (PGK1) RefSeqGene on Chromosome X from position 4644 to 5159). The sequence for the human EFla promoter is shown in SEQ ID NO: 7; this 1188 bp sequence is available at GeneBank Accession No. J04617.1 (human elongation factor EF-1 alpha gene from position 373 to 1560). The WpreMut6 sequence is shown in SEQ ID NO: 8.

The HLA-A2 CAR included: 1) a human CD8 leader sequence (amino acids 1-22; NCBI Ref. Seq. NP_001759.3); 2) a scFv directed against HLA-A2; 3) a linker and a transmembrane domain derived from human CD8 alpha (amino acids 38-206; NCBI Ref. Seq. NP_001759.3); 4) an activation domain of human CD28 (amino acids 180-220; NCBI Ref. Seq. NP 006130.1); and 5) an activation domain of human CD3 zeta (amino acids 52-163; NCBI Ref. Seq. NP_000725.1). The CAR-coding sequence was codon-optimized for Homo sapiens and is shown in SEQ ID NO: 9.

The HLA-A2 CAR amino acid sequence is as followed, wherein the sequence start with the CD8 signal peptide, the VH sequence is underlined, the VL sequence is double-underlined, CD8 hinge and transmembrane domain are in bold, the CD28 domain is in italic and the sequence is ending with the CD3zeta domain:

MALPVTALLL PLALLLHAAR PSQVQLQQSG PELVKPGASV KMSCKASGYT

FTSYHIQWVK QRPGQGLEWI GWIYPGDGST QYNEKFKGKT TLTADKSSST

AYMLLSSLTS EDSAIYFCAR EGTYYAMDYW GQGTSVTVSS GGGGSGGGGS

GGGGSDVLMT QTPLSLPVSL GDQVSISCRS SQSIVHSNGN TYLEWYLQKP

GQSPKLLIYK VSNRFSGVPD RFSGSGSGTD FTLKISRVEA EDLGVYYCFQ

GSHVPRTFGG GTKLEIKRTT TPAPRPPTPA PTIASQPLSL RPEACRPAAG

GAVHTRGLDF ACDIYIWAPL AGTCGVLLLS LVITLYCRSX RSRLLHSDYM

NMTPRRPGPT RKHYQPYAPP RDFAAYRSRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR (SEQ ID NO: 10).

Cloning of HLA-A2 CAR Expression Cassettes into Lentiviral Vectors

The four expression cassettes containing the HLA-A2 CAR were produced using a classical 4-plasmid lentiviral system. Briefly, HEK293T cells were transfected with: (i) the transfer vector (pTX135 or pTX136 or pTX137 or pTX138); (ii) a plasmid expressing HIV-1 gagpol (pMDLgpRRE); (iii) a plasmid expressing HIV-1 rev (pRSV.Rev); and (iv) a plasmid expressing VSV-G, the envelope glycoprotein of the vesicular stomatitis virus (pMD2.G). Twenty-four hours post-transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted, and frozen at -80°C for long-term storage.

The infectious titers were obtained after transduction of a cell line (Jurkat T cells) with a serial dilution of viral supernatants. After 3 days, transduction efficiency was evaluated after Treg labeling with a dextramer specific for the HLA-A2 ScFv and coupled to an Allophycocyanin (APC) fluorophore.

Transduction of Human Tregs with Lentiviral Vectors

The following protocol was used to transduce CD4⁺CD25⁺CD127^lowCD45RA⁺ human Tregs with the TX135, TX136, TX137 and TX138 lentiviral vectors. Briefly, day 3 post-isolation and activation, 750 mΐ of culture medium was removed from each well and 1 x 10⁷ TU/ml of viral vector was added per well. After 6 hours at 37°C, 750 mΐ of fresh medium was added to each well and each well was homogenized, transferred in a tube and centrifuged for 5 minutes at 380g. Supernatants were removed and each pellet was suspended in fresh medium (X-vivol5) supplemented with 1000 U/ml of IL-2 before being seeded onto a new plate.

Fluorescent cell analysis was performed with MACSQUANT analyzer 10 + MACSQuantify software 2.8. RESULTS

Evaluation of CAR Expression Using Dextramer Labeling

Primary Treg cells (CD4⁺CD25⁺CD45RA⁺), sorted from healthy volunteer leukapheresis, were activated with anti-CD3/anti-CD28-coated beads in a 1:1 Treg:Beads ratio and seeded in PL24 at 0.5 x 10⁶ cell per ml before being transduced 3 days later with the different lentiviral vectors described above. As shown in FIG. 1, comparable levels of transduction among the lentiviral constructs were observed in the primary Treg cells.

Although the transduction efficiency was comparable among the four different constructs, the intensity of expression of the CAR on the cell surface was highly variable, with the following ranking: PGK < PGK + Wpremut6 < EFla < EFla Wpremut6 (FIG. 2).

Evaluation of Treg Phenotype Seven Days after Transduction

At day 9 of the culture (7 days after transduction), Treg markers were analyzed to ensure that transduction did not impact the Treg phenotype. The data in FIG. 3 show that the transduced cells displayed a robust Treg phenotype which remained stable after 9 days of post-transduction culturing. All Treg markers, CD4, CD25, FOXP3, Helios, CD127, and CTLA-4, were comparable in expression level between un-transduced and transduced cells.

Evaluation of CAR Activation

After one cycle of expansion, activation of the HLA-A2 CAR was evaluated in the transduced cells. More specifically, we assessed whether HLA-A2 CAR Tregs could be activated through the TCR (as a positive control) and through the CAR by co-incubation with PBMCs from HLA-A2 positive donors. PBMCs from HLA-A2 negative donors were used as a negative control.

After 24 hours of co-incubation, the cell surface expression of CD69 (a surrogate activation marker) was analyzed on Treg cells (FIG. 4). In all the conditions, the activation of HLA-A2 CAR Tregs co-cultured with HLA-A2 negative PBMCs was comparable to un-stimulated Tregs (FIG. 4, Panel B). By contrast, a strong activation was observed for TX135-transduced Tregs stimulated with HLA-A2 dextramers or HLA-A2 positive PBMCs (FIG. 4, Panels A and B). The level of CAR-mediated activation using the strong HLA-A2 dextramer was comparable to Treg stimulation through the TCR (anti-CD3/CD28 beads).

On the contrary, Treg cells expressing the CAR under the PGK promoter (TX135) showed a 3.7-fold increase in CD69 expression following activation by HLA-A2 positive PBMCs, while the increase in CD69 expression was less than 2-fold for Treg cells expressing the CAR under the EFla promoter, namely, pTX137 and pTX138 (FIG. 4, Panel B).

In conclusion, we successfully designed four expression cassettes allowing low to high CAR expression (pTX135<pTX136<pTX137<pTX138). The CAR dependent activation of Tregs ranged from low to high efficiency as followed: pTX138<pTX137<pTX136<pTX135. Thus, the lowest expressed CAR construct, pTX135 (with the PGK promoter and without WpreMut6) allowed the highest CAR-dependent activation in Tregs.

Example 2: CAR Expression Cassettes Using FOXP3- Derived Promoters

This example describes a study aimed to develop CAR expression cassettes with new FOXP3- derived promoters. We evaluated the strengths of these promoters on CAR expression in Tregs by monitoring the cell surface CAR density. Because the endogenous FOXP3 promoter activity can be promoted by interleukin-2 (IL-2) present in the Treg environment (Zorn et al., Blood (2006) 108(5): 1571 -9), the cell surface CAR density was evaluated in Treg cells cultured with different concentrations of IL-2.

We designed four promoters derived from the endogenous core promoter of the human FOXP3 gene. The expression cassettes were carried on lentiviral vectors. We evaluated the strengths of the promoters on expression of the HLA-A2 CAR in Tregs by monitoring the cell surface CAR density. MA TERIALS AND METHODS

The materials and reagents used in the study are shown in Table 3 below.

Table 3 Materials and Reagents

Design and Cloning of FOXP3 Promoters The human FOXP3 gene sequence was obtained from the NCBI server: Homo sapiens chromosome X, GRCh38.pl2 Primary Assembly (NCBI Reference Sequence: NC_000023.11). Numerous transcription factors have been described to interact with the promoter core of the FOXP3 gene, especially the region from -900 nucleotide to +170 nucleotide of the transcription start site (FIG. 5, Panel A). But it is possible that upstream sequences contain enhancer elements and thus also play a key role in transcription regulation. Due to the size limit inherent to the lentiviral genome size, we limited the promoter size to no more than 2kb. Four promoters were designed. Each of them was cloned in a lentiviral backbone to drive the expression of the HLA-A2 CAR (FIG. 5, Panel B). As a control, the classical phosphoglycerate kinase (PGK) promoter was used (pTX300 plasmid).

The four FOXP3 promoters were:

(i) hFXP3.1: - 900 to +3 (nucleotide 49265601 to nucleotide 49264699 of the GRCh38.pl2 Primary Assembly; 903bp; TX319 construct); (11) hFXP3.2: -900 to +172 (nucleotide 49265601 to nucleotide 49264530 of GRCh38.pl2 Primary Assembly; 1072bp; TX320 construct);

(iii) hFXP3.3: -1799 to +3 (nucleotide 49266500 to nucleotide 49264699 of the GRCh38.pl2 Primary Assembly; 1802bp; TX321 construct); and (IV) hFXP3.4: -1799 to +172 (nucleotide 49266500 to nucleotide 49264530 of the

GRCh38.pl2 Primary Assembly; 1971bp; TX322 construct).

Production of Lentiviral Vectors Expressing HLA-A2 CAR

Constructs containing the above five expression cassettes (four with FOXP3 promoters and one with the PGK promoter) were produced using a classical four-plasmid lentiviral system. Briefly, HEK293T cells were transfected with: (i) the transfer vector plasmids (pTX300, pTX319, pTX320, pTX321 or pTX322); (ii) a plasmid expressing HIV-1 gagpol (pMDLgpRRE); (iii) a plasmid expressing HIV-1 rev (pRSV.Rev); and (iv) a plasmid expressing VSV-G, the envelope glycoprotein of the vesicular stomatitis virus (pMD2.G). Twenty-four hours after transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at -80°C for long-term storage. The infectious titers (TU/ml) were obtained after transduction of a cell line (Jurkat T cells) with a serial dilution of viral supernatants and transduction efficiency evaluated after 3 days using a fluorescent dextramer specific for the HLA-A2 ScFv.

Treg Cell Culture Two days before viral transduction (-D2), CD4⁺CD25⁺HLA-A2 Tregs were thawed and cultured in X-Vivol5 medium supplemented with rapamycin and anti-CD3/CD28 beads for activation. On the day of transduction (DO), 5 x 10⁵ Treg cells were transduced with desired lentiviral vector. For that, 5 x 10⁶ TU/ml of viral vector was added per well in a final volume of 200 mΐ. After 6 hours at 37°C, 300 mΐ of fresh medium was added to each well and each well was homogenized, transferred in a tube and centrifuged for 5 minutes at 380g. Supernatants were removed and each pellet was suspended in fresh medium, splitted in three conditions of 200 mΐ supplemented with 50, 200 or 1000 U/ml of IL-2 before being seeded onto a new plate. At days 3, 5, and 7, 50 mΐ of the cell culture was analyzed for CAR MFI; on those days, fresh culture medium supplemented with 50, 200 or 1,000 U/ml IL-2 was given to the remaining cells.

Cell Surface CAR Labeling Cell surface CAR expression was evaluated after cell surface labeling with a HLA-A2 dextramer directly coupled to the Allophycocyanin (APC) fluorophore and flow cytometry analysis using a MACSQUANT analyzer 10 + MACSQuantify software 2.8.

RESULTS

Design of FOXP3 Promoters The core promoter of the human FOXP3 gene has been extensively studied between nucleotides -900 and +200. As shown in FIG. 5, Panel A, numerous binding sites for transcription factors have been described, such as NF-kB, NFAT, API, and FOX03A (Adurthi et al., Sci Rep. (2017) 7:17289; Burchill et al., J Immunol. (2007) 178:280-90; Kitoh et al., Immunity (2009) 31:609-20; Ouyang et al., Nat Immunol. (2009) 11(7):618-27; Zhang et al., J Immunol. (2018) 200(3): 1053-63). Interestingly, three consecutive Stat5-binding domains have been localized between -769 and -729 (Burchill, supra). Stat5 has been described as a major transcription factor in the IL-2 signaling pathway of Tregs.

We designed a first promoter from -900 to +3 (hFXP3.1), which encompasses the Stat5-binding domains. Due to the proximity of the two NF-kB sites between +122 and +149, we designed a second promoter from -900 to +172 (hFXP3.2). Since the upstream sequence of the core promoter has not been studied and could play a key role in FOXP3 transcriptional control, we added to hFXP3.1 and hFXP3.2 an upstream sequence of 899bp, leading respectively to hFXP3.3 (-1799 to +3) and hFXP3.4 (-1799 to +172). HLA-A2 CAR Expression under the Control of FOXP3 Promoters

At day 0, Treg cells were transduced with HLA-A2 CAR-expressing lentiviral vectors harboring the classical PGK (phosphogly cerate kinase) promoter (pTX300), or one of the hFXP3.1 (pTX319), hFXP3.2 (pTX320), hFXP3.3 (pTX321) and hFXP3.4 (pTX322) promoters. One-week post-transduction, the number of HLA-A2 CAR Tregs (%) in the culture and the CAR density at the Treg cell surface (MFI) were evaluated (FIG. 6). As expected, the use of a high viral titer led to an average transduction around 70% (Experiment 1) and the use of a low viral titer led to an average transduction around 40% (Experiment 2). In experiment 1, all FOXP3 promoters were less potent than the PGK promoter. Under all the conditions, the CAR expression (MFI) was not modulated following the culture of Tregs with increasing amounts of IL-2. In experiment 2, we observed a slight sensitivity of FOXP3 promoters to the dose of IL-2, but this observation was not specific since the PGK promoter was also sensitive.

HLA-A2 CAR Density Overtime under the Control of FOXP3 Promoters Next, the CAR density was evaluated overtime (D3, D5 and D7) to monitor the potential accumulation of CAR molecules following the viral transduction. As shown in FIG. 7, using high viral titers (Experiment 1), the CAR expression level under the PGK promoter was shown to increase overtime (average 2-fold increase), independent of the IL-2 concentration. On the contrary, the CAR density didn’t increase for the FOXP3 promoters, except the hFXP3.1 promoter in Tregs cultured with a very low amount (50U) of IL-2 (1.6 fold). Using low viral titers (Experiment 2), some accumulation of the CAR was observed under all conditions but to different extents. While the PGK promoter allowed an average increase of 10-fold of the CAR expression, the FOXP 3 promoters led to an accumulation of only 2 to 3.5-fold overall between day 3 and day 7 post-transduction. Evaluation of the Endogenous FOXP3 Expression in Treg Cells Transduced with a FOXP3 Promoter

At day five post-transduction, the expression level of the endogenous FOXP3 protein was evaluated in un- transduced or TX319-transduced Treg cells cultured in the presence of 50, 200 or 1000 U of IL-2. As shown in FIG. 8, the endogenous FOXP3 expression was highly stable under all conditions, at any IL-2 dose and in either transduced or un-transduced cells. These data indicate that the presence of the artificial FOXP3 promoter in the cells did not impact the ability of the endogenous FOXP3 promoter to drive the expression of the endogenous FOXP3 gene.

Compared to the PGK promoter (TX300 plasmid), we observed that all the artificial FOXP3 promoters were weaker, at either low or high transduction levels. Since the endogenous FOXP3 promoter activity can be promoted by the quantity of IL-2 present in the Treg environment, CAR-Treg cells were cultured with different concentrations of IL-2. Overall, slight increases of expression were observed in correlation with the level of IL-2 for the artificial FOXP3 promoters but not specifically, for the PGK promoter was also slightly sensitive.

In conclusion, this study has allowed us to develop a new family of “very weak” Treg-specific promoters that direct a low level of CAR expression without a strong accumulation overtime, as observed with the “weak” (as opposed to “very weak”) PGK promoter.

Example 3: CAR Expression in Treg cells and Functionality under the Control of Promoters of Various Strengths

This example describes a study aimed to evaluate CD20 CAR expression and functionality under the control of new FOXP3-derived promoters hFXP3.1 and hFXP3.2. We evaluated the strengths of these promoters on CAR expression in Tregs by monitoring the cell surface CAR density. We also evaluated the CAR functionality by monitoring the ligand-independent and ligand-dependent CAR-mediated activation and suppression in Tregs.

MA TE RIALS AND METHODS

The materials and reagents used in the study are shown in Table 4 below. Table 4 Materials and Reagents

Treg, Tconv, B cells and PBMC Isolation

T regulatory (Treg) cells, T conventional (Tconv) cells, and B cells were freshly isolated from huffy coats obtained from heathy volunteer bloods. Briefly, the day after the blood donation, peripheral blood mononuclear cells (PBMC) were isolated from huffy coats by Ficoll gradient centrifugation. Treg cells were isolated following the procedure of the human CD4⁺CD127^lowCD25⁺ Regulatory T Cell Isolation Kit from 400 to 500 x 10⁶ PBL. First, CD25⁺ cells were isolated by column-free, immunomagnetic positive selection using EasySep™ Releasable RapidSpheres™. Then, bound magnetic particles were removed from the EasySep™-isolated CD25⁺ cells, and unwanted non-Tregs were targeted for depletion. The final isolated fraction contained highly purified CD4⁺CD127^lowCD25⁺ cells expressing high levels of FOXP3 and was immediately ready for downstream applications. Autologous CD19⁺CD20⁺ B cells were isolated by immunomagnetic negative selection from 200 x 10⁶ PBL following the procedure of a commercial human B cell isolation kit. After isolation, they were immediately frozen for further used as CD19⁺CD20⁺presenting cells. CD4⁺CD25 Tconv cells were isolated by choosing the optional protocol for the isolation of CD4⁺CD25 responder T cells for use in functional studies in parallel to Tregs.

Design of Different CAR Expression Cassettes

The CAR constructs used in the study are shown below:

TX028: hPGK promoter-CD20 CAR-P2A-GFP TX336: hFXP3.1 promoter-CD20 CAR-P2A-GFP TX337: hFXP3.2 promoter-CD20 CAR-P2A-GFP

The CD20 CAR included: 1) a human CD8 leader sequence (amino acids 1-22; NCBI Ref. Seq. NP_001759.3); 2) a scFv directed against CD20 (B9E9); 3) A streptavidin tag; 4) a linker and a transmembrane domain derived from human CD8 alpha (amino acids 38-206; NCBI Ref. Seq. NP 001759.3); 5) an activation domain of human 4-1BB (amino acids 214-255; NCBI Ref. Seq. NP_001552.2); and 6) an activation domain of human CD3 zeta (amino acids 52-163; NCBI Ref. Seq. NP_000725.1). The CAR-coding sequence was codon-optimized for Homo sapiens. The open reading frame (ORF) of the CAR was in frame with a self-cleaving P2A peptide linker and the ORF of the enhanced green fluorescent protein (GFP). The CD20 CAR-P2A-GFP coding sequence is shown in SEQ ID NO: 24.

The CD20 CAR amino acid sequence is as followed, wherein the sequence starts with a CD8 signal peptide, the VH sequence is underlined, the VL sequence is double-underlined, followed by a streptavidin tag, CD8 hinge and transmembrane domain are in bold, the 4-1BB domain is in italic, the CAR ends with a CD3zeta domain, and finally the CAR is in phase with a P2A-GFP in lowercase:

MALPVTALLL PLALLLHAAR PSQVQLVQSG AELVKPGASV KMSCKASGYT FTSYNMHWVK QTPGQGLEWI GAIYPGNGDT SYNQKFKGKA TLTADKSSST AYMQLSSLTS EDSAVYYCAR AQLRPNYWYF DVWGAGTTVT VSKISGGGGS GGGGSGGGGS GGSSDIVLSQ SPAILSASPG EKVTMTCRAS SSVSYMHWYK QKPGSSPKPW IYATSNLASG VPARFSGSGS GTSYSLTISR VEAEDAATYY CQQWISNPPT FGAGTKLELK SAWSHPQFEK SGMHTTTPAP RPPTPAPTIA SQPLSLRPEA CRPAAGGAVH TRGLDFACDI YIWAPLAGTC GVLLLSLVIT LYCKRG-RX L LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCELTRRVK FSRSADAPAY QQGQNQLYNE LNLGRREEYD VLDKRRGRDP EMGGKPRRKN PQEGLYNELQ KDKMAEAYSE IGMKGERRRG KGHDGLYQGL STATKDTYDA LHMQALPPRa sgsgatnfsl lkqagdveen pgpmvskgee lftgvvpilv eldgdvnghk fsvsgegegd atygkltlkf icttgklpvp wptlvttlty gvqcfsrypd hmkqhdffks ampegyvqer tiffkddgny ktraevkfeg dtlvnrielk gidfkedgni lghkleynyn shnvyimadk qkngikvnfk irhniedgsv qladhyqqnt pigdgpvllp dnhylstqsa lskdpnekrd hmvllefvta agitlgmdel yk (SEQ ID NO: 24).

Cloning of CD20 CAR Expression Cassettes into Lentiviral Vectors

The three expression cassettes containing the CD20 CAR were produced using a classical 4-plasmid lentiviral system. Briefly, HEK293T cells were transfected with: (i) the CD20 CAR-expressing -transfer vector (pTX028, pTX336 or pTX337) or a transfer vector expressing only GFP ; (ii) a plasmid expressing HIV-1 gagpol (pMDLgpRRE); (iii) a plasmid expressing HIV-1 rev (pRSV.Rev); and (iv) a plasmid expressing VSV-G, the envelope glycoprotein of the vesicular stomatitis virus (pMD2.G). Twenty-four hours post-transfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted, and frozen at -80°C for long-term storage.

The infectious titers were obtained after transduction of a cell line (Jurkat T cells) with a serial dilution of viral supernatants. After 3 days, transduction efficiency in Tregs was evaluated by monitoring GFP expression. Culture and Transduction of Human Tregs with Lentiviral Vectors

At day 0, CD4⁺CD25⁺CD127^low human Tregs were isolated and cultured in X-Vivol5 medium complemented with Rapamycin, IL2 and anti-CD3/CD28 beads for activation. On the day of transduction (D2), 5 x 10⁵ Treg cells were transduced with lentiviral vectors. For that, 5 x 10⁶ TU/ml of viral vector was added per well in a final volume of 200 mΐ. After 6 hours at 37°C, 300 mΐ of fresh medium was added to each well and each well was homogenized, transferred in a tube and centrifuged for 5 minutes at 380g. Supernatants were removed and each pellet was suspended in fresh medium. At day 9, 50 mΐ of the cell culture was analyzed for transduction efficiency (% GFP+) and CAR expression (% Prot-L+ and MFI). Fluorescent cell analysis was performed with MACSQUANT analyzer 10 + MACSQuantify software 2.8.

TCR and CAR Activation Assays

At Day 8 post-isolation, the activation assay was performed. Briefly 5x10⁴ Treg cells were seeded in U bottom PL96 alone or in presence of anti-CD3/anti-CD28 coated beads (in a 1 to 1 Treg to beads ratio), or in presence of freshly thawed autologous B cells (in a 1:1 Treg to B cell ratio) in a 200 mΐ final volume. After 24 hours at 37°C, 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry.

Suppression Assay of Tconv Proliferation

At day 8, Treg cells were recovered, counted and activated either through the TCR using anti-CD3/anti-CD28 coated beads (in a 1:1 Treg to beads ratio), or through the CAR using autologous B cells, freshly thawed (in a 1:1 Treg to beads ratio) or kept without activation to evaluate their spontaneous suppressive activity. In parallel, allogeneic Tconv were thawed, stained with Dye 450 and activated with anti-CD3/anti-CD28 coated beads (in a Tconv to beads ratio of 3:1). The following day, beads were removed from Tconv culture before their coculture with un-activated or activated Treg cells. At day 3, cells were harvested, and proliferation of Tconvs was assessed by flow cytometry through determination of Dye 450 dilution. The percentage of inhibition of Tconv proliferation was calculated as followed:

% of Tconv proliferation in presence of CAR-Treg x 100

100 -

% of Tconv proliferation in absence of CAR-Treg

RESULTS Evaluation of Transduction Efficiency and CAR Expression Level

Primary Treg cells (CD4⁺CD25⁺CD127^low) were isolated and activated with anti- CD3/CD28 beads in a 1:1 Treg to bead ratio. After two days, Treg cells were transduced with the lentiviral vectors described above. At day 9 post-isolation, transduction efficiency was determined by assessing the percentage of GFP positive cells (FIG. 9), and CAR expression was monitored by assessing recombinant protein L (Prot-L) labeling, an immunoglobulin kappa light chain-binding protein capable to bind the CD20-CAR. As shown in FIG. 9, the Prot-L mean fluorescence intensity (MFI) representing the number of CARs per cell was decreased by 4 to 5 -fold for CD20-CAR expressed under the control of hFXP3.1 (MFI=26) and hFXP3.2 (MFI=21) promoters compared to hPGK promoter (MFI=102).

This strong decrease of the CAR expression level was not the consequence of a lower transduction efficiency since GFP expression was comparable in all of the experimental conditions.

CAR-Specific Activation As shown in FIG. 10, Panel A, the monitoring of the CD69 cell surface marker allowed us to show a strong reduction of the ligand-independent tonic signaling in Tregs expressing a low level of CD20 CAR (19% for hFXP3 promoters-driven CAR compared to 28% for hPGK-driven CAR with a baseline around 12% for the GFP control condition). Following incubation with anti-CD3/CD28 beads, the Treg activation capacity (% CD69⁺) was comparable for all the conditions (FIG. 10, Panel B). Despite a strong reduction in CD20 CAR expression under the control of hFXP3 promoters, the CAR-specific activation was maintained and even slightly improved (FIG. 10, Panel B). Indeed, the fold of CD69 activation before and after CAR activation using B cells is 1.5-fold for hPGK-driven CAR compared to 1.9 and 1.8-fold for hFXP3-driven CARs.

Evaluation of CAR-Mediated Suppressive Activity in Tregs

The suppressive activity of CAR-Treg cells was evaluated by monitoring the proliferation of Tconv cells co-cultured with CAR-Tregs in the absence or presence of anti-CD3/CD28 beads (TCR activation) or B cells (CAR activation). As expected, the GFP control condition can only be activated through the TCR and not using B cells (no CAR expressed). Interestingly, the spontaneous suppressive activity of the CD20 CAR under the control of the hPGK promoter (TX028) was too strong to highlight a TCR- or CAR-mediated suppressive activity (FIG. 11, top right panel). By contrast, a strong decrease of expression of the same CAR using hFXP3.1 and hFXP3.2 promoters allowed the observation of a potent TCR and CAR-mediated suppressive activity (FIG. 11, bottom panels).

In conclusion, the use of hFXP3 promoters to decrease the expression of a CAR in Tregs can strongly improve its functionality.

Example 4: hFOXP3 Promoters Are Active as Weak Promoters in Cell Types Other Than Tregs

This example describes a study aimed to evaluate IL23R CAR expression and functionality under the control of new QAPi-derived promoter hFXP3.1. We evaluated the strengths of this promoters on CAR expression in T effector cells by monitoring the cell surface CAR expression. MA TERIALS AND METHODS

Except for CAR constructs, the Materials and Methods are the same as those described in Example 2. Here Jurkat-Lucia-NFAT cells were transduced. This reporter cell line is derived from the immortalized human T lymphocyte Jurkat cell. Design of Different CAR Expression Cassettes

The CAR constructs used in the study are shown below:

TX418: hPGK promoter-IL23R CAR-WpreMut6 TX417: hPGK promoter-IL23R CAR TX420: hFXP3.1 promoter- IL23R CAR-WpreMut6 TX419 : hFXP3.1 promoter- IL23R CAR

The IL23 CAR included: 1) a human CD 8 leader sequence; 2) a scFv directed against IL23R; 3) a linker and a transmembrane domain derived from human CD 8 alpha; 4) an activation domain of human CD28; and 5) an activation domain of human CD3 zeta. The CAR-coding sequence was codon- optimized for Homo sapiens and is shown in SEQ ID NO: 53. The amino acid sequence of the IL23R CAR is shown in SEQ ID NO: 54.

Quantification of CAR Expression

The quantification of cell surface CAR expression was performed by labelling the CAR with APC-conjugated protein L, and analyzed using flow cytometry. Median expression levels were determined by gating on the GFP-positive cells (right quadrants). As control served untransduced cells.

RESULTS

In order to test whether the hFOXP3 promoter is active in cell types other than regulatory T-cells, the hFXP3.1 -promoter was tested in the human Jurkat effector T-cell line. Cells were transduced with an IL23R-CAR containing either a hPGK promoter (control) or the hFXP3.1 promoter. Additionally, a construct containing a WPRE was tested.

As shown in FIG. 12, the protein L-based expression analysis showed a significant CAR expression for all constructs, with a pronouncedly lower expression level for the FOXP3 promoter (Median fluorescence intensity of 1600 for hPGK, and 236 for FoxP3, vs. 109 for the Negative control).

The expression level can be further modulated by other transcriptional elements like a WPRE. After the addition of the element the CAR-expression levels increased for the hFXP3.1 promoter by 35% from 236 to 319 (TX419 vs TX420). This is in line from what is found with the hPGK-promoter control 34% increase from 1600 to 2148 (TX417 vs 418).

LIST OF SEQUENCES

The table below lists the amino acid and nucleotide sequences in the present disclosure and their respective SEQ ID NOs (SEQ).

Claims

1. A nucleic acid expression construct, comprising a coding sequence for a protein of interest that is not FOXP3, wherein the coding sequence is operably linked to a promoter derived from a human FOXP3 gene, said promoter comprising nucleotide -769 to nucleotide -729 (SEQ ID NO: 5) of a human FOXP3 gene.

2. The nucleic acid expression construct of claim 1, wherein the promoter comprises nucleotide -900 to nucleotide +3 (SEQ ID NO: 1), nucleotide -900 to nucleotide +172 (SEQ ID NO: 2), nucleotide -1,799 to nucleotide +3 (SEQ ID NO: 3), or nucleotide -1,799 to nucleotide +172 (SEQ ID NO: 4) of the human FOXP3 gene.

3. The nucleic acid expression construct of claim 1 or 2, wherein the construct is a lentiviral construct, an adenoviral construct, an adeno-associated viral construct, a plasmid, a DNA construct, or an RNA construct.

4. The nucleic acid expression construct of any one of the preceding claims, wherein the protein of interest is a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR) or a T-cell receptor (TCR).

5. The nucleic acid expression construct of claim 4, wherein the CAR or TCR is specific for (i) an autoantigen, (ii) a B cell antigen optionally selected from CD 19 and CD20,

(iii) an allogeneic HLA class I molecule, wherein the class I molecule is optionally HLA-A2, (iv) an antigen involved in a disease, or (v) an antigen present or expressed in a specific tissue or organ, or present or expressed at a site of inflammation.

6. A genetically engineered mammalian cell comprising the nucleic acid expression construct of any one of the preceding claims.

7. The genetically engineered cell of claim 6, wherein the cell is a lymphoid cell, a lymphoid progenitor cell, a mesenchymal stem cell, a hematopoietic stem cell, an induced pluripotent stem cell, or an embryonic stem cell.

8. The genetically engineered cell of claim 7, wherein the cell is a T effector (Teff) cell.

9. The genetically engineered cell of claim 7, wherein the cell is a regulatory T (Treg) cell.

10. The genetically engineered cell of any one of claims 6-9, wherein the cell comprises a null mutation in a gene selected from a T cell receptor alpha or beta chain gene, a HLA Class I or II gene, a HLA Class II regulator gene, optionally selected from RFXANK, RFX5, RFXAP (RFX subunits), and CIITA, a transporter associated with antigen processing, a minor histocompatibility antigen gene, and a b2 microglobulin (B2M) gene.

11. The genetically engineered cell of any one of claims 6-10, wherein the cell is a human cell.

12. The genetically engineered cell of claim 11, wherein the cell comprises a suicide gene optionally selected from a HSV-TK gene, a cytosine deaminase gene, a nitroreductase gene, a cytochrome P450 gene, or a caspase-9 gene.

13. A method of treating a patient in need of immunosuppression, comprising administering to the patient a genetically engineered cell of any one of claims 9-12.

14. Use of a genetically engineered cell of any one of claims 9-12 in the manufacture of a medicament in treating a patient in need of immunosuppression.

15. A genetically engineered cell of any one of claims 9-12 for use in treating a patient in need of immunosuppression.

16. The method of claim 13, use of claim 14, or genetically engineered cell for use of claim 15, wherein the patient has a disease or disorder selected from the group consisting of an inflammatory disease or condition, an autoimmune disease or condition, an allergic disease or condition, or an organ transplantation condition, or has received or will receive tissue transplantation.

17. A method of treating cancer or an infectious disease in a patient, comprising administering to the patient a genetically engineered cell of any one of claims 8 and 10 12

18. Use of a genetically engineered cell of any one of claims 8 and 10-12 in the manufacture of a medicament in treating cancer or an infectious disease in a patient.

19. A genetically engineered cell of any one of claims 8 and 10-12 for use in treating cancer or an infectious disease in a patient.

20. The method, use, or genetically engineered cell for use of any one of claims 13-19, wherein the patient is a human.